Project Summary/Abstract We are interested in how ethanol initiates behavioral plasticity. Ethanol inebriation alters the brain, inducing short-term physiological and pathological changes including ethanol tolerance, preference, and reward. These alterations are a foundation for ethanol?s longer-term effects including continued drinking in the face of negative consequences, dysphoria, and relapse. Ethanol?s direct molecular targets, effects on gene transcription, and effects on patterns of neural activity likely combine in complex ways, and are not understood well enough to construct predictive models of ethanol?s action. Binge-like ethanol intake is one of the most reliable predictors of later developing alcohol use disorders. Drosophila, like mammals, become incoordinated and eventually sedated with higher ethanol doses. Acute binge-like ethanol intake causes the development of ethanol tolerance, one of the simplest forms of behavioral plasticity. Our goal is to understand this simple form of ethanol-induced plasticity in as great a detail as possible, in order to uncover fundamental principles of ethanol action in the brain. Current knowledge of tolerance is surprisingly sparse, and how the parts fit together remains unknown. We propose to use Drosophila to understand how ethanol tolerance is coded in the brain. The main goal of this proposal is to determine the brain circuitry for ethanol tolerance. The second goal is to map the sites of plasticity in the tolerance circuitry. Drosophila is exceptionally well-positioned to achieve these goals. Recent advances have made identifying and manipulating small sets of neurons reproducibly in Drosophila much easier and faster than in other model organisms. Furthermore, cell-type specific transcriptomics now works well in Drosophila, allowing us to sample how groups of tolerance neurons respond to alcohol. Drosophila transgenic technology permits easy manipulation of gene expression in these cells to accurately test gene function in tolerance development. Our long term goal is to identify mechanisms of ethanol action that are conserved to humans that may contribute to the development of alcohol use disorders.